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Efficient production method of gamma-aminobutyric acid

A double arginine and glutamic acid decarboxylase technology, which is applied in the field of efficient production of γ-aminobutyric acid, can solve problems such as the difficulty of correct folding and effectively carry out, and achieve the effect of reducing the cost of separation and purification and simplifying the separation and purification procedure

Active Publication Date: 2015-08-12
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Glutamate decarboxylase is a homohexameric protein. Previous research in the laboratory has shown that the Sec system signal peptide PelB cannot secrete active glutamate decarboxylase outside the cell, possibly due to correct folding in the periplasmic space. Difficult to carry out effectively

Method used

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  • Efficient production method of gamma-aminobutyric acid
  • Efficient production method of gamma-aminobutyric acid
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Experimental program
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Embodiment 1

[0021] The construction of embodiment 1 recombinant bacteria

[0022] 1) Genomic DNA was extracted from Escherichia coli E.coli w3110 as a template for PCR amplification, and the primer for amplifying the signal peptide torA gene was: 5'-CGC CATATG ATGAACAATAACGATCTCTTTCAGGC-3' and 5'-CAT CCATGG CCGCTTGCGCCGCAGTC-3', the primer for amplifying the glutamic acid decarboxylase gene gadB is: 5'-CAT CCATGG ATAAGAAGCAAGTAACG-3' and 5'-CC CTCGAG TCAGGTATGTTTAAAAGCTGTT-3'.

[0023] 2) Digest the PCR product of the torA gene with restriction endonucleases NdeI and NcoI, digest the PCR product of the gadB gene with restriction endonucleases NcoI and XhoI, and treat the vector pET20b(+) with NdeI and XhoI. After the digestion product was purified, T4 ligase was ligated overnight at 22 degrees Celsius, transformed into E.coli JM109, and the correct transformant was screened. For the obtained correct transformant, extract the plasmid and transform E.coli BL21(DE3)

Embodiment 2

[0024] Example 2 The extracellular glutamic acid decarboxylase SDS-PAGE and glutamic acid decarboxylase enzyme activity detection of recombinant bacteria at shake flask level

[0025] 1) Shake flask horizontal fermentation of recombinant bacteria

[0026] a) Medium: The seed medium is LB medium (1L): tryptone 10g, yeast extract 5g, NaCl10g, pH adjusted to 7.0; fermentation medium is TB medium (1L): tryptone 12g, yeast extract Extract 24g, Glycerin 5g, K 2 HPO 4 14.6g, NaH 2 PO 4 2H 2 O3.6g, glycine 7.5g, NaCl5g.

[0027] b) Cultivation method: The seeds cultured overnight at 37° C. and 200 rpm were transferred to TB medium at an inoculum size of 5%, and cultivated at 37° C. and 200 rpm for 36 hours.

[0028] c) Induction condition: Induce OD 600 The value is 1.0 and the IPTG concentration is 0.7 mM.

[0029] 2) SDS-PAGE checks the expression of glutamic acid decarboxylase in the cell and outside the cell, with empty load as the control ( figure 1 Among them, 1 and 2 ...

Embodiment 3

[0031] Embodiment 3 Recombinant bacteria fermenter horizontal fermentation

[0032]1) The seed medium is LB medium (1L): tryptone 10g, yeast extract 5g, NaCl10g, pH adjusted to 7.0; fermentation medium is TB medium (1L): tryptone 30g, yeast extract 20g , glycerol 8g, Na 2 SO 4 2.0g, (NH 4 ) 2 SO 4 2.5g, (NH 4 ) 2 -H-citrate1.0g,K 2 HPO 4 14.6g,NaH 2 PO 4 2H 2 O3.6g, MgSO 4 ·7H 2 O2.0g, thiamine100mg; feed medium (1L): yeast extract 50g, yeast extract 50g, glycerin 500g, MgSO 4 ·7H 2 O3.4g; induction (1L): lactose 200g.

[0033] 2) Culture method: when the initial glycerol in the medium is exhausted, that is, when the dissolved oxygen in the fermenter rises, grow at a specific growth rate of 0.12h -1 Exponential flow plus feed medium; to be OD 600 When it reached 50, the feeding medium was changed from exponential feeding to constant feeding, with a flow acceleration rate of 12mL / h, and lactose was fed at a speed of 0.8g / L / h for induction, and the induction ...

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Abstract

The invention discloses an efficient production method of gamma-aminobutyric acid, and belongs to the field of fermentation engineering. In the provided method, a twin-arginine signal peptide gene (torA) and a glutamic acid decarboxylase gene (gadB) are fused and transferred to a escherichia coil BL21(DE3) host so as to obtain a recombinant bacterium pET20b(+)-torA-gadB / E.coli BL21(DE3) that can secret and express glutamic acid decarboxylase. The extracellular glutamic acid decarboxylase activity level of the recombinant bacterium in a shaking bottle can reach 5.11 U / mL, and 15.82 U / mL in a fermentation tank. By using the system and a substrate (sodium glutamate monohydrate), the output of gamma-aminobutyric acid can reach 203.7 g / L.

Description

technical field [0001] The invention relates to a method for efficiently producing gamma-aminobutyric acid, in particular to a method for efficiently producing gamma-aminobutyric acid by using a genetically engineered bacterium that secretes and expresses glutamic acid decarboxylase, and belongs to the field of fermentation engineering. Background technique [0002] γ-aminobutyric acid (GABA for short), also known as 4-aminobutyric acid and aminobutyric acid, is a non-protein amino acid that exists widely in nature and has important physiological functions. In the human body, GABA is one of the three major inhibitory neurotransmitters in the central nervous system. It has physiological effects such as relieving anxiety, lowering blood pressure, and calming sleep, so it is widely used in medical care. The production of GABA by fermentation mainly uses the glutamate decarboxylase (glutamate decarboxylase, GAD, EC 4.1.1.15) of the microorganism itself to catalyze the decarboxyl...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N9/88C12P13/00C12R1/19
CPCC12N1/20C12N9/88C12N15/70C12P13/005C12Y401/01015C12N1/205C12R2001/19
Inventor 王小元赵安琪胡晓清李烨
Owner JIANGNAN UNIV
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